I. Adenylated RNA-DNA repair by Aptx The frequency of ribonucleotide incorporation into DNA in cycling cells exceeds other known DNA damage combined. A major ribonucleotide excision repair (RER) pathway is initiated by RNase H2 incision on the 5 side of a DNA embedded ribonucleotide. We hypothesized that abundant incised ribonucleotide intermediates arising during RER might also be substrates for otherwise unanticipated reactions or side reactions during commonly occurring DNA transactions. One example of this is DNA ligation. Eukaryotic ATP-dependent DNA ligases catalyze nick sealing during DNA replication and repair with a three step mechanism involving active site adenylation of the ligase, adenylate transfer to the DNA 5&#8242;-phosphate, and DNA nick sealing with release of AMP. However, when DNA ligases engage nicked DNA substrates with preexisting DNA damage, for instance an RNA-DNA junction from RER, ligase can undergo abortive ligation where the enzyme dissociates prematurely from its substrate following DNA adenylation. In this case, rather than sealing a DNA nick to finalize DNA replication or repair, ligase catalyzes the addition of a bulky AMP adduct to the 5&#8242; terminus of the nicked substrate. We established that RNA-DNA junctions arising from RER are indeed subject to abortive ligation by human DNA ligases 1 and 3 in vitro. RER intermediates trigger ligation failure and production of compounded DNA damage in the form of adenylated RNA-DNA lesions. Aprataxin (Aptx) is a member of the histidine triad (HIT) family of nucleoside hydrolases, and catalyzes direct reversal of DNA 5&#8242;-adenylation resulting from abortive ligation. Aptx therefore may be critical for genome stability in cells undergoing RER. Consistent with a roles for Aprataxins in metabolizing RNA derived damage, budding yeast cells with elevated ribonucleotides but lack the Aptx homolog deficient cells (hnt3&#916;) display marked defects in cellular proliferation, activation of the S-phase DNA damage checkpoint, and are sensitive to the replication inhibitor hydroxyl urea (HU). These phenotypes are relieved in an rnh201&#916; background, supporting a model where Aptx (Hnt3p) is required for efficient repair adenylated RNA-DNA junctions that arise from RNase H2 incision followed by abortive ligase metabolism. Human and yeast aprataxins all efficiently catalyze reversal of adenylated RNA-DNA in vitro, and a series of X-ray crystal structures of hAptx bound to adenylated RNA-DNA structures further establishes the molecular basis for adenylated RNA-DNA damage processing. The mechanism involves structure-specific A-form RNA-DNA recognition of the adenylated RNA 5&#8242; terminus, and encirclement of the lesion pyrophosphate linkage to facilitate direct reversal of the adenylated RNA-DNA lesion. Notably high-resolution structural analysis of a disease causing Aptx mutation (K197Q) linked to Ataxia with Oculomotor Apraxia 1 (AOA1) distorts the Aptx RNA-DNA lesion binding pocket II. Repair of ribonucleotide linked Top2cc by Tdp2 Normally, the eukaryotic type II topoisomerases (Top2&#945; and Top2&#946;) regulate DNA topology by employing a dsDNA cleavage and religation cycle involving transient formation of Top2-DNA cleavage-complexes (Top2cc). Top2 catalytic intermediates are characterized by the topoisomerase covalently linked to the DNA 5&#8242;-terminus by an active site tyrosine residue (Fig. 1A). However, aberrant DNA structure or targeted chemotherapeutic disruption of the Top2 reaction can generate Top2cc, protein-DNA crosslinks that block transcription and/or collapse DNA replication forks. Interestingly, ribonucleotides stimulate the Top2&#945; and Top2&#946; DNA cleavage reactions, and therefore are potentially toxic to the Top2 reaction cycle by producing increased Top2RNA-DNA cleavage complex. We have been studying a major pathway for repair of Top2cc that is present vertebrates, but absent in lower eukaryotes including yeasts. This pathway involves direct reversal of Top2-DNA phosphortyrosyl linkages by tyrosyl DNA phosphodiesterase 2 (Tdp2). Tdp2 (aka Ttrap/Eap2/VPg unlinkase) also catalyzes reversal of protein-RNA covalent linkages during RNA replication of picornaviruses (e.g poliovirus and rhinovirus), suggestive of broader RNA repair functions for Tdp2. Recently we established Tdp2 also efficiently processes phosphotyrosine covalently adducted to 5&#8242; ribonucleotides. To understand the molecular basis for RNA-protein processing by Tdp2, we solved a high-resolution structure of Tdp2 bound to a 5-ribonucleotide containing substrate. This work defines a mechanism through which RNA containing substrates are engaged by Tdp2 in a manner similar to that of DNA-only substrates. Overall these results are suggestive that genomic instability triggered by ribonucleotides in DNA might also be mediated by formation of Top2RNA-DNA cleavage complexes, and production of DNA single strand and double strand breaks. In an RNA-DNA repair role, emerging results suggest Tdp2 might also protect from RNA-triggered events in vivo. III. Mechanisms of DNA end processing by Ctp1Ctip/Sae2/Mre11/Rad50/Nbs1 DNA double strand breaks (DSBs) generated by clastogen exposures including ionizing radiation and topoisomerase poisons can sever entire chromosomes, thereby contributing to genomic instability and carcinogenesis. Error-free DSB repair of adducted DNA strand breaks by homologous recombination (HR) is initiated by the Mre11/Rad50/Nbs1 (MRN) complex. Ctp1CtIP/Sae2 collaborates with the Mre11-Rad50-Nbs1 (MRN) nuclease to modulate end processing, but the functional roles for Ctp1 remain unclear. We have established that Ctp1 harbors DNA-binding and bridging activities, but is not a nuclease. Our Ctp1 X-ray structures, small angle X-ray scattering (SAXS), and biophysical analysis define the salient features of Ctp1 architecture: an N-terminal interlocking tetrameric helical dimer of dimers domain (THDD), and an extended central intrinsically disordered region (IDR) linked to conserved C-terminal DNA-binding RHR motifs. The THDD, IDR and RHR regions are all required to support Ctp1 DNA bridging activity in vitro, and THDD or RHR disruption confers sensitivity of fission yeast to DNA damaging agents. Together, our results establish functional roles for tetrameric Ctp1 in the binding and coordination of DSB repair intermediates, and suggest that disruption of CtIP DNA binding activity by truncating mutations underlies CtIP-linked neurodegenerative Seckel and Jawad syndromes.